Soluble analyte detection and amplification

ABSTRACT

The present invention relates to a method of detection of a compound of interest that is present at low levels in a sample. In particular, the present invention relates to a method of detection of a compound of interest in solution by a nucleic acid-labelled binding construct, separation of the unbound nucleic acid-labelled binding construct, and the detection of the bound nucleic acid-labelled binding construct in the solution phase. The present invention is particularly adaptable to be used in conjunction with a nucleic acid amplification reaction for detecting the presence or absence of the nucleic acid portion of binding construct in a sample indicating the presence or absence of the compound of interest.

FIELD OF INVENTION

The present invention relates generally to the field of compounddetection, and particularly to methods for compound detection and a kitfor compound detection.

BACKGROUND OF THE INVENTION

The detection of immune responses to pathological organisms or thedetection of pathogen-related proteins or other antigens in the serum ofpatients has benefited greatly from immunoassay development over thelast 15 years. In one form of immunoassay, monoclonal or polyclonalantibodies that recognize the immunoglobulins of another species areused. These reagents, known as anti-species antibodies are labelled,typically with a fluorochrome or enzyme, and used to detect antigenbinding by immunoglobulins found in immune serum. In another form ofimmunoassay, known as the sandwich assay, antibodies directed againstpathogen proteins are used to capture antigen from, for example,patients' serum or cerebrospinal fluid (CSF), that is then detected bythe binding of another, labelled antibody directed against the sameantigen. All of these assays, however, are limited by the sensitivity ofdetection of the bound immunoglobulin and require fairly largeconcentrations, on a molar basis, of labelled reagent.

More recent assays utilize nucleic acid amplification methods, such asthe polymerase chain reaction (PCR) for DNA amplification, to detectvery low levels of nucleic acid. Nucleic acid amplification methods canallow the detection of pathological agents in serum or in theenvironment at levels well below that of immunoassay detection. Suchtechniques, however, are often very sensitive to contamination from theenvironment and require prior knowledge of the nucleic acid sequence ofinterest in order to identify a portion of the nucleic acid and amplifyit for detection. Nucleic acid amplification requires that there benucleic acid present to amplify and detect and is, therefore, of no orlimited utility when the compound of interest is a protein,carbohydrate, or other non-nucleic acid molecule. Further improvementsin the effectiveness and sensitivity of compound detection in a sampleare desirable, and the present invention addresses the existing problemsand provides related benefits.

SUMMARY OF THE INVENTION

Throughout this application various publications are referenced. Thedisclosures of these publications are hereby incorporated by reference,in their entirety, in this application. Citations of these documents arenot intended as an admission that any of them are pertinent prior art.All statements as to the date or representation as to the contents ofthese documents is based on the information available to the applicantand does not constitute any admission as to the correctness of the datesor contents of these documents.

The present invention relates to a method of detection of a compound ofinterest that can be present at low levels in a sample. In particular,the present invention relates to a method of detection of a compound ofinterest in solution by a nucleic acid-labelled binding construct thatbinds the compound of interest, separation of the unbound nucleicacid-labelled binding construct, and the detection of the bound nucleicacid-labelled binding construct in the solution phase. The presentinvention is particularly adaptable to be used in conjunction with anucleic acid amplification method for detecting the presence or absenceof the nucleic acid portion of the binding construct in a sample, thusindicating the presence or absence of the compound of interest.

The present invention recognizes that compound detection methods can bemade more sensitive and thereby more effective by separation from thesolution any excess or unbound binding construct that is not bound tothe compound of interest, thus increasing the proportion of true“positive” signal to false “positive” signal. The present inventionfurther provides a versatile detection system that does not requiresolid-phase capture or detection of the compound of interest. Where thecompound of interest includes a non-nucleic acid molecule such as apeptide or protein, a method of the present invention further providesthe advantage of not requiring prior knowledge of the nucleic acidsequence encoding the peptide or protein of interest in order toidentify it and to use DNA amplification for its detection.

A first aspect of the present invention is a method for detecting acompound of interest in a sample, including the use of a bindingconstruct. The binding construct includes a recognition portion whichrecognizes and binds the compound of interest, and a nucleic acidportion. When the binding construct is mixed with the sample, therecognition portion binds with the compound of interest and formsconstruct-compound complexes. The present invention also includessurfaces bearing one or more accessible binding targets capable ofbinding to the recognition portion of the binding construct. When thesurfaces are contacted with the mixture of the sample and bindingconstruct, the accessible binding targets of the surfaces bind with anyexcess or unbound binding construct to form construct-surface complexes.After sufficient incubation, the construct-surface complexes and anyunbound or excess surfaces are separated from the mixture leaving behindthe construct-compound complexes in solution. After separation, thesolution is analysed in order to detect the presence or absence of thenucleic acid portion of the binding construct, wherein the presence ofthe nucleic acid portion of the binding construct indicates the presenceof the compound of interest in the sample.

A second aspect of the present invention is a method for increasing thesensitivity of solution-phase detection of a compound of interest in asample. The method includes providing a sample suspected of containingthe compound of interest; providing a binding construct including arecognition portion capable of binding the compound of interest, and anucleic acid portion; and contacting the sample with the bindingconstruct for a period of time sufficient to permit the recognitionportion to bind any compound of interest in the sample, thus formingconstruct-compound complexes in solution. The method further includesproviding one or more surfaces that bears one or more accessible bindingtarget capable of binding to the recognition portion, and contacting thesurface with the solution for a period of time sufficient for theaccessible binding target to bind the recognition portion of any bindingconstruct not bound to the compound of interest, thereby formingconstruct-surface complexes. The construct-surface complexes areseparated from the solution, leaving the construct-compound complexes inthe solution. The presence or absence of the nucleic acid portion of thebinding construct is detected in the solution. The separation of theconstruct-surface complexes from the solution results in a separation ofsubstantially all binding constructs not bound to a compound of interestand in an increased sensitivity of detection of the compound ofinterest, and the presence of the nucleic acid portion of the bindingconstruct indicates the presence of the compound of interest in thesample.

A third aspect of the present invention is a kit for detecting acompound of interest including a binding construct comprising arecognition portion which recognizes and binds the compound of interest,and a nucleic acid portion. The kit of the present invention alsoincludes one or more surfaces bearing one or more accessible bindingtargets capable of binding to the recognition portion of the bindingconstruct. The kit of the present invention can also optionally includea nucleic acid amplification primer pair, wherein each primer of theprimer pair hybridizes to its complementary sequence at the 3′ end of atarget nucleic acid sequence of the nucleic acid portion of the bindingconstruct.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a plurality of binding constructs, each including arecognition portion and a nucleic acid portion (see Example I).

FIG. 2 depicts a plurality of binding constructs, some of which haverecognized and bound a compound of interest to their recognition portionto form a construct-compound complex (see Example I).

FIG. 3 depicts the surfaces bearing accessible binding targets, whichare recognized by and are bound to the recognition portion of thebinding constructs forming construct-surface complexes (see Example I).

FIG. 4 depicts separation of the construct-surface complexes from theconstruct-compound complexes (see Example I).

FIG. 5 depicts results of Example II: The Mopep2 particles were capableof binding to and separating monoclonal antibody 12D5 (12D.5 Mab)horseradish peroxidase (HRPO) conjugates from solution, thus preventingthe 12D.5 Mab HRPO conjugate from binding to Mopep2-coated wells in adose-dependent manner. Particles coated with bovine serum albumin (BSA)were unable to inhibit 12D.5 Mab HRPO conjugate from binding toMopep2-coated wells. OD, optical density.

FIG. 6 depicts the results obtained in Example IV, using a method of theinvention to detect a compound of interest wherein the nucleic acidportion of the binding construct is amplified by PCR. This experimentdemonstrated the ability of the binding construct Fab-DNA (Mab12D.5/pUC19 construct) to bind to the compound of interest, the freeantigen (bacterial recombinant fragment of OMPE or rOMPE), thus formingconstruct-compound complexes in solution. Surfaces (magnetic particles)bearing accessible binding targets (Mopep2 peptides) bound any Fab-DNAnot bound to the compound of interest, and a magnet was used to separatethe surfaces, leaving the construct-compound complexes (Fab-DNA bound toantigen) in solution with the nucleic acid portion (pUC19) of thebinding construct available for nucleic acid amplification. Polymerasechain reaction amplification resulted in a detectable 2.6 kilobase DNAfragment in the following samples: 790 picogram, 395 picogram, 198picogram, 98 picogram, and 12 picogram. Very fine 2.6 kilobase bandswere also observed in samples of 49 picogram and 24 picogram.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention recognizes that compound detection methods can bemade more sensitive and thereby more effective by increasing theproportion of true “positive” signal to false “positive signal”. Thepresent invention further recognizes the versatility of a detectionsystem that does not require solid-phase detection of the compound ofinterest, and further recognizes the desirability of benefiting fromsignal amplification methods (such as nucleic acid amplification) evenin the detection of a compound of interest that does not include anucleic acid.

As a non-limiting introduction to the breadth of the present invention,the present invention includes several general and useful aspects,including:

-   -   1) A method for detecting a compound of interest in a sample,        including the steps of: providing a binding construct including        a recognition portion which recognizes and binds the compound of        interest, and a nucleic acid portion; mixing the binding        construct with the sample to form construct-compound complexes;        providing one or more surface, wherein the surface bears one or        more accessible binding targets capable of recognizing and        binding to the recognition portion of the binding construct;        introducing the surface to the mixture of the binding construct        and the sample in order for the surface to form        construct-surface complexes with any unbound binding constructs;        separating the construct-surface complexes from the mixture        leaving behind the construct-compound complexes; detecting the        presence or absence of the nucleic acid portion of the binding        construct, wherein the presence of the nucleic acid portion of        the binding construct indicates the presence of the compound of        interest in the sample.    -   2) A method for increasing the sensitivity of solution-phase        detection of a compound of interest, including the steps of:        providing a sample suspected of containing the compound of        interest; providing a binding construct that includes a        recognition portion capable of binding the compound of interest,        and a nucleic acid portion; contacting the sample with the        binding construct for a period of time sufficient to permit the        recognition portion to bind any compound of interest in the        sample, thus forming construct-compound complexes in solution;        providing one or more surfaces that bears one or more accessible        binding target capable of binding to the recognition portion;        contacting the surface with the solution for a period of time        sufficient for the accessible binding target to bind the        recognition portion of any binding construct not bound to the        compound of interest, thereby forming construct-surface        complexes; separating the construct-surface complexes from the        solution, leaving the construct-compound complexes in the        solution; and detecting the presence or absence of the nucleic        acid portion of the binding construct in the solution, wherein        the separation of the construct-surface complexes from the        solution results in a separation of substantially all binding        constructs not bound to a compound of interest and in an        increased sensitivity of detection of the compound of interest,        and wherein the presence of the nucleic acid portion of the        binding construct indicates the presence of the compound of        interest in the sample.    -   3) A kit for detecting a compound of interest comprising: a        binding construct comprising a recognition portion which        recognizes and binds the compound of interest, and a nucleic        acid portion; and one or more surfaces, wherein the surfaces        bear one or more accessible binding targets known to be capable        of binding to the recognition portion of the binding construct.        The kit includes, optionally, a nucleic acid primer pair,        wherein the wherein each primer of the primer pair hybridizes to        its complementary sequence at the 3′ end of a target nucleic        acid sequence of the nucleic acid portion of the binding        construct.

The present invention relates to a sensitive method for the detection ofa compound of interest present in a sample, such as a biological fluid,a biological extract, or an environmental sample, by means of a nucleicacid-labelled binding construct which is capable of recognizing andbinding the compound of interest. This compound detection method iscalled soluble analyte detection and amplification. The presentinvention provides a method for detection of a compound of interest thatis particularly adaptable for use with samples wherein the compound ofinterest is present in the sample in low levels. “Sensitivity” can bedefined as the proportion of true positives detected by a systemdesigned to discriminate between two categories, known conventionally aspositive and negative. The present invention provides enhanced detectionsensitivity because it is possible to provide even excess amounts of thebinding construct, and then separate from the sample any unbound (thatis to say, not bound to a compound of interest) or excess bindingconstruct (detection of which would lead to a false “positive” signal),thus leaving in solution only the binding construct that is bound to acompound of interest in a construct-compound complex available fordetection as a true “positive” signal. Thus, detection sensitivity isproportional to the efficiency of separation of unbound or excessbinding construct from the solution. The present invention is alsogenerally more versatile because detection occurs in the solution phaseand is not restricted to solid phase detection. Furthermore, only onebinding entity (the binding construct) is required to recognize and bindthe compound of interest, thus avoiding the problems associated withmethods using two binding entities, for example, the problems of sterichindrance or potential loss of sensitivity from conformational changesinduced by the binding of a first antibody, such as may occur forexample, in a two-antibody sandwich assay.

Further objectives and advantages of the present invention will becomeapparent as the description proceeds and when taken in conjunction withthe accompanying drawings. To gain a full appreciation of the scope ofthe present invention, it will be further recognized that variousaspects of the present invention can be combined to make desirableembodiments of the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the manufacture or laboratory procedures described beloware well known and commonly employed in the art. The technical termsused herein have their ordinary meaning in the art that they are used,as exemplified by a variety of technical dictionaries. Where a term isprovided in the singular, the inventor also contemplates the plural ofthat term. The nomenclature used herein and the procedures describedbelow are those well known and commonly employed in the art. Where thereare discrepancies in terms and definitions used in references that areincorporated by reference, the terms used in this application shall havethe definitions given herein. Other technical terms used herein havetheir ordinary meaning in the art that they are used, as exemplified bya variety of technical dictionaries (for example, Chambers Dictionary ofScience and Technology, Peter M. B. Walker (editor), Chambers HarrapPublishers, Ltd., Edinburgh, UK, 1999, 1325 pp.). The inventors do notintend to be limited to a mechanism or mode of action. Reference theretois provided for illustrative purposes only.

I. Method for Detecting a Compound of Interest in a Sample

The first method of the present invention includes a sensitive methodfor detection of a compound of interest in a sample that is capable ofdetecting the presence of very low quantities of the compound ofinterest in the sample. The first method of the present inventioninvolves the recognition of a compound of interest by a nucleicacid-labelled binding construct, separation of the unbound nucleicacid-labelled binding construct by means of one or more surfaces bearingone or more binding targets, and detecting in solution the presence orabsence of the nucleic acid portion of the binding construct wherein thepresence of the nucleic acid portion of the binding construct indicatesthe presence of the compound of interest in the sample. The samplesuspected of containing the compound of interest may be, for example, ofentirely natural origin, of entirely non-natural origin (such as ofsynthetic origin), or a combination of natural and non-natural origins.The sample may include whole cells, tissues, organs, biological fluids,extracts, or environmental samples.

The first method of the present invention includes use of a bindingconstruct. The binding constructs are mixed with the sample, allowingthe recognition portion of the binding construct to bind to the compoundof interest to form construct-compound complexes in solution.

The binding construct includes a recognition portion that can recognizeand bind to the compound of interest, and a nucleic acid portion. Therecognition portion can include virtually any molecule or combination ofmolecules capable of recognizing and binding the compound of interest.Such recognition portions can include, without limitation, peptides,polypeptides, antibodies, Fab fragments, nucleic acids, nucleic acidmimics, cell surface antigens, carbohydrates, or combination thereof. Inone embodiment, the recognition portion includes an antibody (natural,modified, or recombinant) or an antibody fragment (such as an Fabfragment or single-chain antibody variable region fragment). In otherembodiments, the recognition portion can be an antigen that binds anantibody, an aptamer that binds a target such as a peptide or smallmolecule, or a receptor that binds a ligand. In one preferredembodiment, the recognition portion binds monovalently to the compoundof interest. In another preferred embodiment, the recognition portionbinds multivalently, for example bivalently and optionallybispecifically, to the compound of interest.

The nucleic acid portion of the binding construct can include anynucleic acid or nucleic acid mimic that is capable of being detected.The nucleic acid that is used as the nucleic acid portion of the bindingconstruct can include any type of nucleic acid, for example DNA or RNA,or a nucleic acid mimic (such as, but not limited to, a peptide nucleicacid), or a combination thereof. The nucleic acid of the invention canbe single-stranded or double-stranded. In one preferred embodiment, thebinding construct of the present invention includes a nucleic acidportion, wherein the sequence of the nucleic acid portion does notinclude a sequence that is expected to be found in the sample, and canthereby be reasonably expected to be less easily contaminated by nucleicacid sequences in the sample, but is, however, designed so as to beeasily detectable. The nucleic acid portion is preferably sufficientlylong enough to be easily detectable by the detection method chosen.

The recognition portion can be attached to the nucleic acid portion byany method, covalently or non-covalently, directly or indirectly, and itwill depend on the nature of a given recognition portion. Non-covalentattachment methods include but are not limited to physical adsorption,electrostatic forces, ionic interactions, hydrogen bonding,hydrophilic-hydrophobic interactions, van der Waals forces, and magneticforces. Where desired, for example, when increased flexibility isneeded, a recognition portion may be indirectly affixed to the nucleicacid portion using a spacer arm. Preferably, the recognition portion isattached to the nucleic acid portion via covalent linkage, or via a highaffinity non-covalent interaction such as that of biotin and avidin.

The first method of the present invention includes the use of one ormore surfaces that bears one or more accessible binding targets that areknown to be recognized by and bind to the recognition portion of thebinding construct. Such surfaces can be any particle capable of beingseparated from a liquid sample, or such surfaces may be non-particulatesurfaces such as planar or non-planar surfaces, or a combinationthereof. The surfaces can be optionally enclosed, for example, in achamber. The binding targets can include any target capable of beingrecognized by and binding to the recognition portion of the bindingconstruct, for example, peptide mimotopes or epitope mimetics forantibody variable regions, whole or partial antigen, nucleic acids, orsugar moieties. The accessible binding target can be attached to thesurface by any method, and it will depend on the nature of a givensurface and binding target.

When the surfaces are introduced to the mixture of the sample andbinding construct, after sufficient incubation, the accessible bindingtargets of the surfaces bind any unbound recognition portions of thebinding construct forming construct-surface complexes. Theconstruct-surface and any excess unbound surfaces are then separatedfrom the construct-compound complexes in solution in the sample, bymethods suitable to the type of surface used, for example, a magnet toseparate magnetic particles, decanting from a planar surface, orseparation by pressure or vacuum, centrifugation, or filtration.Alternatively, any binding construct not bound to a compound of interestin a construct-compound complex can be separated from solution bymethods such as, but not limited to, precipitation, “salting-out”,size-exclusion or filtration, extraction, or phase-separation.

After the separation step, by detecting the presence or absence of thenucleic acid portion of the binding construct, the presence or absenceof the compound of interest in the sample is determined. The presence orabsence of the nucleic acid portion of the binding construct can bedetected by any method that is capable of detecting the presence orabsence of nucleic acid, for example, enzymatic amplification,hybridization, or detection of a label. In one embodiment of the presentinvention, the method can be preferably adapted to detect the presenceor absence of the nucleic acid portion of the binding construct byamplification of the nucleic acid portion, for example, by means of apolymerase chain reaction with appropriate primers. In this embodiment,only a few, and at least only one, construct-compound complex needs toserve as a template for nucleic acid amplification. The amplifiednucleic acid can be measured or detected by any suitable method, forexample, using labelled oligonucleotides or by detecting appropriatebands on polyacrylamide gel electrophoresis.

In one embodiment of the present invention, the sample to be analyzedmay contain two or more compounds of interest. In order to detect two ormore different compounds of interest in a sample, two or more differenttypes of binding constructs, each having a different recognition portioncapable of recognizing a different compound, are provided. Each type ofbinding construct includes a nucleic acid portion unique to the type, inorder to detect the presence or absence of two or more compounds ofinterest in the sample.

II. Method from Increasing the Sensitivity of Solution-Phase Detectionof a Compound of Interest

The second method of the present invention includes a method forincreasing the sensitivity of solution-phase detection of a compound ofinterest. The method is especially suitable for samples that aresuspected to contain a low concentration or low quantities of thecompound of interest.

The second method of the present invention includes the steps of:providing a sample suspected of containing the compound of interest;providing a binding construct that includes a recognition portioncapable of binding the compound of interest, and a nucleic acid portion;contacting the sample with the binding construct for a period of timesufficient to permit the recognition portion to bind any compound ofinterest in the sample, thus forming construct-compound complexes insolution; providing one or more surfaces that bears one or moreaccessible binding target capable of binding to the recognition portion;contacting the surface with the solution for a period of time sufficientfor the accessible binding target to bind the recognition portion of anybinding construct not bound to the compound of interest, thereby formingconstruct-surface complexes; separating the construct-surface complexesfrom the solution, leaving the construct-compound complexes in thesolution; and detecting the presence or absence of the nucleic acidportion of the binding construct in the solution, wherein the separationof the construct-surface complexes from the solution results in aseparation of substantially all binding constructs not bound to acompound of interest and in an increased sensitivity of detection of thecompound of interest, and wherein the presence of the nucleic acidportion of the binding construct indicates the presence of the compoundof interest in the sample.

The second method of the present invention includes the steps ofproviding a sample suspected of containing a compound of interest, andproviding a binding construct. The sample suspected of containing thecompound of interest may be, for example, of entirely natural origin, ofentirely non-natural origin (such as of synthetic origin), or acombination of natural and non-natural origins. The sample may includewhole cells, tissues, organs, biological fluids, extracts, orenvironmental samples. The binding construct includes a recognitionportion that can recognize and bind to the compound of interest, and anucleic acid portion. The sample is contacted with the binding constructfor a period of time sufficient to permit the recognition portion of thebinding construct to bind the compound of interest and form aconstruct-compound complex in solution.

The recognition portion of the binding construct can include virtuallyany molecule or combination of molecules capable of recognizing andbinding the compound of interest. Such recognition portions can include,without limitation, peptides, polypeptides, antibodies, Fab fragments,nucleic acids, nucleic acid mimics, cell surface antigens,carbohydrates, or combination thereof. In one embodiment, therecognition portion includes an antibody (natural, modified, orrecombinant) or an antibody fragment (such as an Fab fragment orsingle-chain antibody variable region fragment). In other embodiments,the recognition portion can be an antigen that binds an antibody, anaptamer that binds a target such as a peptide or small molecule, or areceptor that binds a ligand. In one preferred embodiment, therecognition portion binds monovalently to the compound of interest. Inanother preferred embodiment, the recognition portion bindsmultivalently, for example bivalently and optionally bispecifically, tothe compound of interest.

The nucleic acid portion of the binding construct can include anynucleic acid or nucleic acid mimic that is capable of being detected.The nucleic acid that is used as the nucleic acid portion of the bindingconstruct can include any type of nucleic acid, for example DNA or RNA,or a nucleic acid mimic (such as, but not limited to, a peptide nucleicacid), or a combination thereof. The nucleic acid of the invention canbe single-stranded or double-stranded. In one preferred embodiment, thebinding construct of the present invention includes a nucleic acidportion, wherein the sequence of the nucleic acid portion does notinclude a sequence that is expected to be found in the sample, and canthereby be reasonably expected to be less easily contaminated by nucleicacid sequences in the sample, but is, however, designed so as to beeasily detectable. The nucleic acid portion is preferably sufficientlylong enough to be easily detectable by the detection method chosen.

The recognition portion can be attached to the nucleic acid portion byany method, covalently or non-covalently, directly or indirectly, and itwill depend on the nature of a given recognition portion. Non-covalentattachment methods include but are not limited to physical adsorption,electrostatic forces, ionic interactions, hydrogen bonding,hydrophilic-hydrophobic interactions, van der Waals forces, and magneticforces. Where desired, for example, when increased flexibility isneeded, a recognition portion may be indirectly affixed to the nucleicacid portion using a spacer arm. Preferably, the recognition portion isattached to the nucleic acid portion via covalent linkage, or via a highaffinity non-covalent interaction such as that of biotin and avidin.

The second method of the present invention includes providing one ormore surfaces bearing one or more accessible binding targets that areknown to be recognized by and bind to the recognition portion of thebinding construct. Such surfaces can be any particle capable of beingseparated from a liquid sample, or such surfaces may be non-particulatesurfaces such as planar or non-planar surfaces, or a combinationthereof. The surfaces can be optionally enclosed, for example, enclosedin a chamber. The binding targets can include any target capable ofbeing recognized by and binding to the recognition portion of thebinding construct, for example, peptide mimotopes or epitope mimeticsfor antibody variable regions, whole or partial antigen, nucleic acids,or sugar moieties. The accessible binding target can be attached to thesurface by any method, and it will depend on the nature of a givensurface and binding target.

The surfaces are contacted with the solution for a period of timesufficient for the accessible binding target to bind the recognitionportion of any binding construct not bound to a compound of interest,thus forming construct-surface complexes. The construct-surfacecomplexes and any excess unbound surfaces are separated from thesolution, leaving behind the construct-compound complexes. Separationmay be by any method suitable to the type of surface used, for example,a magnet to separate magnetic particles, decanting from a planarsurface, or separation by pressure or vacuum, centrifugation, orfiltration. Alternatively, any binding construct not bound to a compoundof interest in a construct-compound complex can be separated fromsolution by methods such as, but not limited to, precipitation,“salting-out”, size-exclusion or filtration, extraction, orphase-separation. Separation of the construct-surface complexes and anyexcess unbound surfaces results in a separation of substantially allbinding constructs not bound to a compound of interest. Most preferably,all binding constructs not bound to a compound of interest are separatedfrom solution. Separation of the construct-surface complexes and anyexcess unbound surfaces results in a decrease in “false” positive signalgenerated by binding complexes not bound to a compound of interest, andthus in an increased sensitivity of detection of the compound ofinterest relative to an assay wherein unbound or excess bindingconstructs is not separated.

After the separation step, by detecting the presence or absence of thenucleic acid portion of the binding construct, the presence or absenceof the compound of interest in the sample is determined. The presence orabsence of the nucleic acid portion of the binding construct can bedetected by any method that is capable of detecting the presence orabsence of nucleic acid, for example, enzymatic amplification,hybridization, or detection of a label. In one embodiment of the presentinvention, the method can be preferably adapted to detect the presenceor absence of the nucleic acid portion of the binding construct byamplification of the nucleic acid portion, for example, by means of apolymerase chain reaction with appropriate primers. In this embodiment,only a few, and at least only one, construct-compound complex needs toserve as a template for nucleic acid amplification. The amplifiednucleic acid can be measured or detected by any suitable method, forexample, using labelled oligonucleotides or by detecting appropriatebands on polyacrylamide gel electrophoresis.

In one embodiment of the present invention, the sample to be analyzedmay contain two or more compounds of interest. In order to detect two ormore different compounds of interest in a sample, two or more differenttypes of binding constructs, each having a different recognition portioncapable of recognizing a different compound, are provided. Each type ofbinding construct includes a nucleic acid portion unique to the type, inorder to detect the presence or absence of two or more compounds ofinterest in the sample.

III. Kit for Detection of a Compound of Interest

The present invention also includes a kit used for the detection of acompound of interest in a sample that is capable of detecting thepresence of very low quantities of the compound of interest in thesample.

The kit of the present invention includes a binding construct. Thebinding construct includes a recognition portion that can recognize andbind to the compound of interest, and a nucleic acid portion. Therecognition portion can include virtually any molecule or combination ofmolecules capable of recognizing and binding the compound of interest.Such recognition portions can include, without limitation, peptides,polypeptides, antibodies, Fab fragments, nucleic acids, nucleic acidmimics, cell surface antigens, carbohydrates, or combination thereof. Inone embodiment, the recognition portion includes an antibody (natural,modified, or recombinant) or an antibody fragment (such as an Fabfragment or single-chain antibody variable region fragment). In otherembodiments, the recognition portion can be an antigen that binds anantibody, an aptamer that binds a target such as a peptide or smallmolecule, or a receptor that binds a ligand. In one preferredembodiment, the recognition portion binds monovalently to the compoundof interest. In another preferred embodiment, the recognition portionbinds multivalently, for example bivalently and optionallybispecifically, to the compound of interest.

The nucleic acid portion of the binding construct can include anynucleic acid or nucleic acid mimic that is capable of being detected.The nucleic acid that is used as the nucleic acid portion of the bindingconstruct can include any type of nucleic acid, for example DNA or RNA,or a nucleic acid mimic (such as, but not limited to, a peptide nucleicacid), or a combination thereof. The nucleic acid of the invention canbe single-stranded or double-stranded. In one preferred embodiment, thebinding construct of the present invention includes a nucleic acidportion, wherein the sequence of the nucleic acid portion does notinclude a sequence that is expected to be found in the sample, and canthereby be reasonably expected to be less easily contaminated by nucleicacid sequences in the sample, but is, however, designed so as to beeasily detectable. The nucleic acid portion is preferably sufficientlylong enough to be easily detectable by the detection method chosen.

The recognition portion can be attached to the nucleic acid portion byany method, covalently or non-covalently, directly or indirectly, and itwill depend on the nature of a given recognition portion. Non-covalentattachment methods include but are not limited to physical adsorption,electrostatic forces, ionic interactions, hydrogen bonding,hydrophilic-hydrophobic interactions, van der Waals forces, and magneticforces. Where desired, for example, when increased flexibility isneeded, a recognition portion may be indirectly affixed to the nucleicacid portion using a spacer arm. Preferably, the recognition portion isattached to the nucleic acid portion via covalent linkage, or via a highaffinity non-covalent interaction such as that of biotin and avidin.

The kit of the present invention also includes one or more surfaces thatbears one or more accessible binding targets that are known to berecognized by and bind to the recognition portion of the bindingconstruct. Such surfaces can be any particle capable of being separatedfrom a liquid sample, or such surfaces may be non-particulate surfacessuch as planar or non-planar surfaces, or a combination thereof. Thesurfaces can be optionally enclosed, for example, in a chamber. Thebinding targets can include any target capable of being recognized byand binding to the recognition portion of the binding construct, forexample, peptide mimotopes or epitope mimetics for antibody variableregions, whole or partial antigen, nucleic acids, or sugar moieties. Theaccessible binding target can be attached to the surface by any method,and it will depend on the nature of a given surface and binding target.

In embodiments wherein the nucleic acid portion of the binding constructis detected by nucleic acid amplification, the kit can optionallyinclude a nucleic acid amplification primer pair, for example, a PCRprimer pair wherein each primer of the primer pair hybridizes to itscomplementary sequence at the 3′ end of a target nucleic acid sequenceof the nucleic acid portion. The kit can further optionally includeenzymes for nucleic acid amplification, such as Taq polymerase or RNAreverse transcriptase. In embodiments wherein the nucleic acid portionof the binding construct is detected by nucleic acid hybridization, thekit may contain one or more hybridization probes, such asoligonucleotides labelled with a detectable label. In embodimentsincluding amplification of a signal, the kit may optionally include thereagents needed for signal amplification, such as enzymes or substrates.Kits of the invention can optionally include reagents for directdetection of the nucleic acid portion of the binding construct, such asmolecular beacons or an appropriate antibody. The kit can optionallyinclude means for separation of construct-surface complexes and anyunbound surfaces, for example a magnet for embodiments wherein thesurface is a magnetic particle, a filter for embodiments wherein thesurface is a filterable particulate, or a pipette for embodimentswherein the surface is the walls of a tube.

Optionally, the kit can include instructions for the use of the kit.Such instructions may be in any suitable form, such as a brochure,leaflet, pamphlet, booklet, or audiovisual materials. Preferably theinstructions are sufficiently detailed to permit a user of the kit tosuccessfully use the kit to detect a compound of interest in a sample.Such instructions may include, for example, instructions for mixingreagents, manipulating components of the kit, proper handling of asample, guidance in safety measures and in interpreting results, andtrouble-shooting instructions.

The Compound of Interest

The methods and kits of the present invention may be used to detectvarious classes of compounds of interest. The methods are particularlysuitable to the detection of compounds of interest that are suspected tobe present in low amounts or low concentrations in a sample. Compoundsof interest can include, but are not limited to, nucleic acids,peptides, proteins, glycoproteins, lipoproteins, lectins, antibodies,enzymes, and receptors; carbohydrates (monosaccharides,oligosaccharides, and polysaccharides) and glycosylated molecules;lipids, fats, and lipidated molecules; and whole or partial antigens.Compounds of interest may be small molecules (for example, ligands for areceptor, drugs of abuse, inorganic ions, metals, or chelates,metabolites, chemical intermediates, or natural products). Compounds ofinterest may be monomers, oligomers, or polymers; they can also bemulti-molecular assemblies (for example, amyloid beta protofibrils, thedystrophin-glycoprotein assembly, proteosomes, chaperone proteins, orfragments of cell walls or cell membranes). Compounds of interest may beof completely natural origin, completely artificial origin, or acombination of both (such as a compound of natural origin that ischemically or physically modified).

The Sample

The methods of the invention may be applied to any suitable sample thatis suspected of containing the compound of interest. The sample may beof entirely natural origin, of entirely non-natural origin (such as ofsynthetic origin), or a combination of natural and non-natural origins.A sample may include whole cells (such as prokaryotic cells, bacterialcells, eukaryotic cells, plant cells, fungal cells, or cells frommulti-cellular organisms including invertebrates, vertebrates, mammals,and humans), tissues, organs, or biological fluids (such as, but notlimited to, blood, serum, plasma, urine, semen, and cerebrospinalfluid). A sample may be an extract made from biological materials, suchas from prokaryotes, bacteria, eukaryotes, plants, fungi, multi-cellularorganisms or animals, invertebrates, vertebrates, mammals, non-humanmammals, and humans. A sample may be an extract made from wholeorganisms or portions of organisms, cells, organs, tissues, fluids,whole cultures or portions of cultures, or environmental samples orportions thereof. A sample may need minimal preparation (for example,collection into a suitable container) for use in a method of the presentinvention, or more extensive preparation (such as, but not limited toremoval, inactivation, or blocking of undesirable material orcontaminants, filtration, size selection, affinity purification, celllysis or tissue digestion, concentration, or dilution). The sample maybe in any phase (solid, liquid, or gaseous), in solution or insuspension, as long as it may be treated (such as by mechanicaldisruption, lysis, heating, addition of a solvent, or suspension agent)to permit detection of the construct-compound complex in solution. Forexample, a sample may be a soil sample, which may be suspended in anaqueous buffer and optionally filtered to remove undesired particulatesbefore introduction of the binding construct.

The Binding Construct

The present invention includes a binding construct. The bindingconstruct includes a recognition portion that can recognize and bind tothe compound of interest, and a nucleic acid portion. The recognitionportion can be virtually any molecule or combination of moleculescapable of recognizing and binding the compound of interest. Suchrecognition portions can include, without limitation, peptides,polypeptides, mimotopes, antibodies, Fab fragments, nucleic acids,nucleic acid mimics, aptamers, cell surface antigen, carbohydrates,small molecules (such as a small molecule antigen or a ligand for areceptor), inorganic ions or chelates, or a combination thereof.Particularly preferred is a recognition portion capable of monovalentbinding to the compound of interest.

In one embodiment, the recognition portion is an antibody, for example,human or other mammalian IgG, IgG₁, IgG₂, IgG₃, IgG₄, IgM, IgA, SigA, orIgE, or avian IgY, and in one preferable embodiment, the recognitionportion is an antibody fragment. Preparation of antibodies and antibodyfragments against a compound of interest is well known in the art. Thesetechniques are described in, for example, Antibodies, A LaboratoryManual, (Harlow and Lane) Cold Spring Harbor Laboratory Press (1988),and updated in Using Antibodies, A Laboratory Manual, (Harlow and Lane)Cold Spring Harbor Laboratory Press (1999). Where the recognitionportion is an antibody or antibody fragment, it can be natural (such asan immunoglobulin isolated from serum), modified (such as a reduced ordeglycosylated antibody), recombinant (such as an antibody produced byphage display), or a combination thereof.

The recognition portion can be a natural, modified, or recombinantantibody binding fragment, such as an Fab fragment, or such as asingle-chain antibody variable region fragment or ScFv in which therecombinant variable regions of an immunoglobulin's light and heavychain domains are connected by a linker sequence (Pantoliano et al.,(1991) Biochemistry, 30:10117-10125). In one embodiment, the recognitionportion of the binding construct includes an Fab fragment, capable ofmonovalent binding to the compound of interest. Fab fragments againstthe compound of interest can be produced when antibodies against thecompound of interest are cleaved by reduction or enzymatic cleavage ofthe disulfide bridge that holds the heavy chains together, converting anantibody into two separate Fab fragments, each capable of recognizingand binding the compound of interest, and bearing a reactive sulfhydrylor thiol group that can be attached to other molecules, for example, thenucleic acid portion of the binding construct. In one embodiment, a Fabfragment can be directly covalently attached to a nucleic acid portionthrough the free sulfhydryl of the Fab fragment, thus forming thebinding construct. In another embodiment, the Fab fragment can beindirectly covalently attached to the nucleic acid portion of thebinding construct through a bi-functional linker capable of specificallybinding both said Fab fragment and said nucleic acid portion forming thebinding construct. Alternatively, the recognition portion of the presentinvention can also be attached to the nucleic acid portion vianon-covalent means, for example, via an avidin-biotin interaction, azinc-polyhistidine interaction, an antibody-antigen interaction, anaptamer-peptide interaction, or via other polypeptides capable ofbinding nucleic acids such as zinc-binding polypeptide domain.

The recognition portion (for example, peptides, mimotopes, antibodies,or aptamers) can include natural molecules, artificial molecules, fusionor chimeric molecules, molecules developed by random or non-randomcombinatorial synthesis (Dooley and Houghten (1993) Life Sci., 52:1509-1517; Kramer et al. (1993) Peptide Res., 6:314-319; Folgori et al.(1994) EMBO J., 13:2236-2243; Smith & Petrenko (1997) Chem. Rev.,97:391-410) or by directed evolution methods such as yeast two-hybridsystems, protein fragment complementation assay, phage display, ribosomedisplay, yeast surface display, and bacterial surface display techniques(Crameri and Suter (1993) Gene, 137:69-75; Meola et al. (1995) J.Immunol., 154:3162-3172; Georgiou et al. (1997) Nature Biotechnol.,15:29-34; Mössner and Plückthun (2001) Chimia, 55:324; B. K. Kay, J.Winter, and J. McCafferty (editors), “Phage Display of Peptides andproteins: A Laboratory Manual”, Academic Press, Inc., San Diego, 1996,344 pp.), or a combination thereof. The recognition portion can beselected for its ability to recognize and bind the compound of interestby any suitable means known in the art, for example, by affinityselection, affinity purification, iterative panning, or surface plasmonresonance technology (Fägerstam et al. (1991) J. Mol. Recognition,3:208-214; Houshmand et al. (1999) Anal. Biochem., 268:363-370).

The recognition portion of the binding construct may bind to thecompound of interest in a monovalent or in a multivalent fashion. In onepreferred embodiment, the binding of the recognition portion of thebinding construct to the compound of interest is monovalent. Examples ofmonovalent binding include, but are not limited to, a Fab fragment thatmonovalently binds to an antigen, a receptor molecule that monovalentlybinds its ligand, and an aptamer that monovalently binds a peptide. Incertain other embodiments, the binding of the recognition portion of thebinding construct to the compound of interest may preferably bemultivalent, for example, bivalent or trivalent. Multivalency may bedesirable, for example, to increase the avidity of the binding betweenthe binding construct and the compound of interest. In some embodiments,the binding of the recognition portion of the binding construct to thecompound of interest may be both bivalent and bispecific (that is, therecognition portion may bind to and recognize two separate specificbinding sites of the compound of interest). An example of a bivalent,monospecific binding is a dimeric antibody fragment or diabody (Holligeret al. (1993) Proc. Natl. Acad. Sci. USA, 90:6444-6448) that is designedto bind monospecifically. An example of a bivalent, bispecific antibodyis a diabody that is designed to bind to two distinct binding sites ofthe compound of interest.

The nucleic acid portion of the binding construct can include anynucleic acid or nucleic acid mimic that is capable of being detected.The nucleic acid that is used as the nucleic acid portion of the bindingconstruct can include any type of nucleic acid, for example DNA or RNA,or a nucleic acid mimic (such as, but not limited to, a peptide nucleicacid), or a combination thereof. In this respect, the nucleic acidportion of the binding construct acts as a marker for detection, forexample, for detection through nucleic acid amplification, nucleic acidhybridization, enzymatic signal amplification, detection of a label, ora combination of these detection methods. The nucleic acid of theinvention can be single-stranded or double-stranded.

In one preferred embodiment, the binding construct of the presentinvention includes a nucleic acid portion, wherein the sequence of thenucleic acid portion does not include a sequence that is expected to befound in the sample, and can thereby be reasonably expected to be lesseasily contaminated by nucleic acid sequences in the sample, but is,however, designed so as to be easily detectable. For example, where thesample is a serum sample from a mammal, the sequence of the nucleic acidportion can include a nucleic acid sequence believed to occur only inhigher plants, and thus unlikely to be found in mammalian serum. Inanother preferred embodiment, the binding construct of the presentinvention includes a nucleic acid portion, wherein the sequence of thenucleic acid portion does not include a sequence that is believed tooccur in nature. In other embodiments, the nucleic acid portion of theinvention can include artificially derived sequences (such as sequencesarrived at by random or non-random combinatorial approaches) orrepetitive sequences, for example, repetitive sequences that may becomplementary to a single hybridization primer or probe. In otherembodiments, the nucleic acid portion of the invention can includemultiple, repetitive nucleic acid portions attached in series or inparallel to the recognition portion.

The nucleic acid portion is preferably sufficiently long enough to beeasily detectable by the detection method chosen. Where detection of thenucleic acid portion includes nucleic acid amplification, the nucleicacid portion preferably includes a single-strand length of between about50 to about 5000 nucleotides, or between about 100 to about 4000nucleotides, or between about 200 and about 3000 nucleotides. However,the nucleic acid portion may include any number of nucleotides that issuitable to the chosen method of amplification of the nucleic acidportion, for example PCR or reverse-transcriptase PCR. Where detectionof the nucleic acid portion does not include nucleic acid amplification,the nucleic acid portion preferably includes a single strand length ofbetween about 4 nucleotides to about 5000 nucleotides, or between about20 nucleotides to about 4000 nucleotides, or between about 100nucleotides to about 3000 nucleotides. However, the nucleic acid portionmay include any number of nucleotides that is suitable to the chosenmethod of detection of the nucleic acid portion.

The recognition portion can be attached to the nucleic acid portion byany method, covalently or non-covalently, directly or indirectly,depending on the nature of a given recognition portion and nucleic acidportion. Such attaching methods can be, for example, covalentcross-linking as well as non-covalent linking methods such as are knownin the art (see, for example, R. P. Haugland, “Handbook of FluorescentProbes and Research Products”, 9^(th) edition, J. Gregory (editor),Molecular Probes, Inc., Eugene, Oreg., USA, 2002, 966 pp.; Seitz andKohler (2001), Chemistry, 7:3911-3925; and Pierce Technical Handbook,Pierce Biotechnology, Inc., 1994, Rockford, Ill.). Where desired, forexample, when increased flexibility is needed, a recognition portion maybe affixed to the nucleic acid portion using a spacer arm. (Keyes et al.(1997) Biophys. J., 72:282-90; Hustedt et al. (1995) Biochemistry,34:4369-4375; and Pierce Technical Handbook, Pierce Biotechnology, Inc.,1994, Rockford, Ill.). In one embodiment, the recognition portion isattached to the nucleic acid portion via covalent linkage. Covalentmeans are well known in the art and may include, for example, the use ofreactive groups, chemical modification or activation, photoactivatedcross-linking, or bifunctional or trifunctional cross-linking agents(Pierce Technical Handbook, Pierce Biotechnology, Inc., 1994, Rockford,Ill.). In another embodiment, the recognition portion is attached to thenucleic acid portion via non-covalent means. Non-covalent means include,but are not limited, to physical adsorption, electrostatic forces, ionicinteractions, hydrogen bonding, hydrophilic-hydrophobic interactions,van der Waals forces, and magnetic forces. A combination of covalent andnon-covalent attachment means may be used. For example, the nucleic acidportion (or repetitive multiples of the nucleic acid portion) may bebiotinylated and non-covalently attached to a multivalent avidin moietythat is covalently cross-linked to the recognition portion.

The Surfaces and Accessible Binding Targets

The present invention also includes one or more surfaces that bears oneor more accessible binding targets that are known to be recognized byand bind to the recognition portion of the binding construct. Suchsurfaces can be particulate surfaces or non-particulate surfaces, andcan be made of any suitable material, such as, but not limited to,plastics, polymers, ceramics, glass, silica compounds, modified silicacompounds, fluorocarbons, metals or metal oxides, sorbents, resins,biological materials (for example, polypeptides and carbohydrates), or acombination thereof. Particulate surfaces can be any particle capable ofbeing separated from a liquid sample, for example, magnetic particles,polymeric particles, glass particles, silica particles, ceramicparticles, and the like. Particulate surfaces can be any shape includingspherical, non-spherical, symmetric, asymmetric, or irregular; they canbe of uniform or non-uniform size. Particulate surfaces can take anysuitable form, for example, powders, beads, fibers, macromolecularaggregates, nanoparticles, or nanotubes. Particulate surfaces may beoptionally enclosed in a chamber, such as in a reusable or a disposablecartridge, cassette, or insert. Non-particulate surfaces include but arenot limited to planar or non-planar surfaces (for example, the sides ofa tube or a well), non-porous films or membranes, porous films ormembranes, fibers, fillers, meshes, grids, filters, matrices, gels, or acombination thereof.

The accessible binding targets of the present invention can be anybinding target capable of being recognized by and binding to therecognition portion of the binding construct, for example, nucleicacids, peptide mimotopes or epitope mimetics for antibody variableregions (Geysen et al. (1986) Mol. Immunol., 23:709-715), proteins,glycoproteins, lipoproteins, lectins, antibodies, enzymes, receptors;carbohydrates (monosaccharides, oligosaccharides, and polysaccharides)and glycosylated molecules; lipids, fats, and lipidated molecules; andwhole or partial antigens; small molecules (for example, ligands for areceptor, drugs of abuse, inorganic ions or chelates, metabolites,chemical intermediates, or natural products). Accessible binding targetsmay be monomers, oligomers, polymers; they can also be multi-molecularassemblies. Accessible binding targets may be of completely naturalorigin, completely artificial origin, or a combination of both (such asa compound of natural origin that is chemically modified).

The accessible binding target can be attached to the surface by anymethod, covalent or non-covalent, directly or indirectly, depending onthe nature of a given surface and accessible binding target. Theaccessible binding target can be attached to the surfaces by covalentbonds, for example, through the reaction of a chemically reactive groupof the accessible binding target with a chemically reactive group of thesurface, using appropriate reagents. For example, binding targetsbearing primary amines can be linked to amine-binding supports to form asecondary amine using sodium cyanoborohydride; binding targets bearingcarbohydrates can be linked to surfaces bearing a free hydrazide groupto form a stable hydrazone bond. In another example,1-ethyl-3[3-dimethylaminopropyl]-carbodiimide hydrochloride (EDC) can beused to link carboxyl groups to surfaces bearing primary amines. Theaccessible binding target can be attached to the surface by non-covalentmeans, including, but not limited, to physical adsorption, electrostaticforces, ionic interactions, hydrogen bonding, hydrophilic-hydrophobicinteractions, van der Waals forces, and magnetic forces. Non-limitingexamples of non-covalent means include biotin-avidin interactions,protein A or protein G interactions with immunoglobulins, andantigen-antibody interactions.

The accessible binding target may be covalently attached to surfaces,such as, but not limited to, polymeric, ceramic, glass, silica, ororganic surfaces, through a variety of surface chemistries. Surfaces,both particulate and non-particulate, are readily available withcarboxyl, amino, hydroxyl, hydrazide, or chloromethyl functional groups.In one embodiment, where the surface is a magnetic particle, commercialmagnetic stocks may be used, and may be selected for properties allowingthe desired accessible binding target to be attached to the magneticparticles. For example, the commercially available magnetic particlestock Micromod, Nanomag Silica™, NH250 (catalogue number 13-01-252,Micromod Partikeltechnologie GmbH, Rostock-Warnemuende, Germany)contains on its surface a free amino group, which can be reacted withcross linkers, for example, N-succinimidyl(4-iodoacetyl)aminobenzoate(SIAB) orsulfosuccinimidyl-4-(N-maleimindomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC), each of which has an amine-reactive end and asulfhydryl-reactive end. The NSH ester of SIAB can couple to primaryamine containing molecules, and the ensuing reaction is a stable amidelinkage.

Separation

The construct-surface and any excess unbound surfaces are separated fromthe construct-compound complexes in the sample solution using anysuitable method of separation, which can depend on the type of surfaceused. Such methods can include, without limitation, separation bypressure or vacuum, centrifugation, size-exclusion, filtration, or acombination thereof. The following are non-limiting examples ofseparation. Where the surfaces are particulates (such as powders, beads,fibers, macromolecular aggregates, nanoparticles, or nanotubes), theconstruct-surface complexes can be separated by sedimentation,centrifugation, filtration, size-exclusion, or non-covalent attraction(for example, charge or hydrophobicity interaction). Where the surfacesare planar or non-planar non-particulates (such as the walls of amicrotiter well or a tube or vessel), the construct-surface complexescan be separated by decanting or aspiration. Where the surfaces aremagnetic particles, separation of the construct-surface complexes fromthe mixture may be accomplished by application of magnetic force, suchas by exposure to a magnet.

In one embodiment, the surfaces can be enclosed, permanently ortemporarily, in a chamber (such as in a tube, cartridge, column, orcassette), which can facilitate separation of the excess or unboundbinding construct. For example, the mixture containing the sample andbinding construct can be passed through a cartridge or column containingaccessible binding targets bound to surfaces (such as beads, a matrix, agel, or a filter), whereby the solution that is eluted or that passesthrough the cartridge or column is stripped of excess or unbound bindingconstruct.

In some embodiments, “separation” of the construct-surface complexesfrom the mixture need not require physical removal of theconstruct-surface complexes from the solution, where it is sufficient toisolate the construct-surface complexes from the solution-phasedetection step. For example, where the surfaces are magnetic particles,adequate separation of the construct-surface complexes from the mixturemay be accomplished by application of magnetic force, for example, tothe side of a vessel containing the mixture, thus attracting themagnetic particles to the side of the vessel and isolating them from thesolution, allowing an aliquot of the solution to be sampled for thedetection step.

In an alternative embodiment, any binding construct not bound to acompound of interest in a construct-compound complex can be separatedfrom the solution by methods such as, but not limited to, precipitation,“salting-out”, size-exclusion or filtration, extraction, orphase-separation. In this alternative embodiment, the principles of themethods of the invention remain the same, that is to say, (1) the use ofonly a single binding entity (the binding construct); (2) the separationof substantially all binding constructs not bound to a compound ofinterest, resulting in an increased proportion of true “positive” signalto false “positive” signal, and therefore an increased sensitivity ofdetection of the compound of interest; (3) detection in solution phase,and no requirement for solid-phase detection; and (4) no requirement forprior knowledge of a nucleic acid sequence of the compound of interest.

Detection

After the separation step, by detecting the presence or absence of thenucleic acid portion of the binding construct, the presence or absenceof the compound of interest in the sample is indicated. The presence orabsence of the nucleic acid portion of the binding construct can bedetected by any method, not necessarily involving nucleic acidamplification, that is capable of detecting the presence or absence ofnucleic acid, for example, enzymatic amplification, hybridization, ordetection of a label. The detection of the nucleic acid portion of thebinding construct can be accomplished by any method suitable for suchpurpose. These methods are well known in the art and they include,without limitation, amplification of the nucleic acid portion,hybridization of the nucleic acid portion, amplification of a signal,detection of a label, or a combination thereof.

Methods of the present invention can include nucleic acid amplificationof the nucleic acid portion. Preferred embodiments of the presentinvention are capable of detecting low amounts or concentrations of thecompound of interest, and are capable of amplifying and detecting a few,and a minimum of one, construct-compound complexes. In such preferredembodiments, only a few, and a minimum of one, construct-compoundcomplexes need be remain in solution to serve as amplification templatesafter the separation of the construct-surface complexes. The amplifiednucleic acid can be measured or detected by any suitable method, forexample, using labelled oligonucleotides as hybridization probes or bydetecting amplified fragments of the appropriate size by polyacrylamidegel electrophoresis. Amplification of the nucleic acid portion may useany suitable amplification method, such as polymerase chain reactionamplification or reverse transcriptase amplification (Molecular Cloning:A Laboratory Manual, Joseph Sambrook et al., Cold Spring HarborLaboratory, 2001, 999 pp.; Short Protocols in Molecular Biology,Frederick M. Ausubel et al. (editors), John Wiley & Sons, 2002, 1548pp.), rolling circle amplification (Liu et al. (1996), J. Am. Chem.Soc., 118:1587-1594), antisense RNA amplification (Phillips and Eberwine(1996) Methods, 10:283-288), strand displacement amplification (Walkeret al. (1992), Nucleic Acids Res., 20:1691-1696), compositeprimer/strand displacement amplification (U.S. Pat. No. 6,251,639 toKurn, “Methods and compositions for linear isothermal amplification ofpolynucleotide sequences, using a RNA-DNA composite primer”, issued Jun.26, 2001), Q-beta replicase-mediated amplification (Lomeli et al. (1989)Clin. Chem., 35:1826-1831), linked linear amplification (Reyes et al.(2001) Clin. Chem., 47:31-40), self-sustained sequence replication (3SR)(Fahy et al. (1991) Genome Res., 1:25-33), or other nucleic acidamplification methods known in the art (Andras et al. (2001) Mol.Biotechnol., 19:29-44). Another method of detection of the nucleic acidportion of the binding construct can be, for example, by primerextension and detection of the extended nucleic acid.

Hybridization of the nucleic acid portion may be achieved byhybridization of a probe to the nucleic acid portion of the bindingconstruct, followed by detection of the hybridized structure, accordingto methods known in the art. The probe may include DNA, RNA, a nucleicacid mimic (such as, but not limited to, a peptide nucleic acid), or acombination thereof. The probe may include a suitable detectable label,such as, but not limited to, radioisotopes, spin labels, fluorophores(including organic dyes and lanthanide chelates), chromophores, one orboth members of a resonance energy transfer pair, haptens, antigens,antibodies, or enzymes. A label, for example, a fluorophore or a hapten,can also be incorporated directly into the nucleic acid by methods knownin the art followed by the detection of the label. Detection of thenucleic acid may include enzymatic amplification of a signal (such as asignal from a label on a probe), for example by usingperoxidase-tyramide signal amplification (R. P. Haugland, “Handbook ofFluorescent Probes and Research Products”, 9^(th) edition, J. Gregory(editor), Molecular Probes, Inc., Eugene, Oreg., USA, 2002, 966 pp.) oralkaline phosphatase-anti-alkaline phosphatase signal amplification. Thenucleic acid portion of the binding construct can also be detected,directly or after amplification or hybridization, by other methods,including but not limited to molecular beacons (optionally combined withreal time detection) or other resonance energy transfer methods, orimmunological detection (for example, using antibodies that recognizeand capture an amplified DNA sequence in an ELISA-type assay).

When detection of the nucleic acid portion of the binding constructinvolves amplification, a preferred method is amplification of thenucleic acid portion by polymerase chain reaction, according to methodsknown in the art (Molecular Cloning: A Laboratory Manual, JosephSambrook et al., Cold Spring Harbor Laboratory, 2001; Short Protocols inMolecular Biology, Frederick M. Ausubel et al. (editors), John Wiley &Sons, 2002). The capability to detect a compound of interest in a samplecan be significantly increased and broadened by coupling to polymerasechain reaction. PCR allows for enormous amplification capability and isa process where a specific sequence of nucleic acid can be amplifiedmillions of times. This enormous amplification capability is based onthe ability to amplify a specific target sequence of nucleic acidflanked by a set of primers. Once the nucleic acid is amplified, theresult can be detected using any method suitable for such purpose, forexample, using an agarose gel.

EXAMPLES Example I A Non-Limiting Embodiment of a Method for Detectionof a Compound of Interest in a Sample

FIG. 1 depicts a plurality of binding constructs for use in a method ofthe present invention, as described in Example III. Each bindingconstruct 101 includes a recognition portion 102, which recognizes andbinds the compound of interest, and a nucleic acid portion 103. In thisembodiment, the recognition portion 102 preferably includes a Fabfragment, such as a Fab fragment made by cleaving the disulfide bridgeof the heavy chains of a monoclonal antibody against the compound ofinterest. In this embodiment, the nucleic acid portion 103 preferablyincludes DNA. The recognition portion 102 can be attached to the nucleicacid portion 103 through a covalent bond 104, for example, through acovalent bond between the nucleic acid portion 103 and the freesulfhydryl of the Fab fragment of the recognition portion 102.

FIG. 2 shows a number of binding constructs 101 that have been mixedwith a sample containing a compound of interest 201, as described inExample IV. The recognition portion 102 of the binding construct 101 hasrecognized a compound of interest 201 forming a construct-compoundcomplex 202. Also shown in the lowest part of the figure is a bindingconstruct 101, including a recognition portion 102, which has not boundthe compound of interest 201, and a nucleic acid portion 103.

FIG. 3 shows surfaces 301, bearing accessible binding targets 302. Inthis embodiment, the surfaces 301 are magnetic particles and theaccessible binding targets 302 are peptide mimotopes, as described inExample II. The surfaces are introduced to the sample mixture, which cancontain binding constructs 101, which may be bound to a compound ofinterest in a construct-compound complex 201, or not bound to a compoundof interest. The accessible binding target 301 binds to the recognitionportion 102 of any binding construct 101 not bound to a compound ofinterest, forming a construct-surface complex 303.

FIG. 4 shows a magnet 401 being used to separate the construct-surfacecomplexes 303, as described in Example IV. The construct-surfacecomplexes 303 and any excess or unbound surfaces 301 are separated,leaving the construct-compound complexes 202 in solution. Afterseparation, the nucleic acid portion 103 of the binding construct 101included in a construct-compound complex 202 can be detected by anymethod suitable for that purpose. Detecting the presence of the nucleicacid portion 103 of the binding construct 101 indicates the presence ofthe compound of interest 201.

Example II Preparation of the Magnetic Particles Bearing Mimotopes

This example provides one non-limiting embodiment of the presentinvention wherein the surfaces are magnetic particles bearing peptidemimotopes as the accessible binding targets. In this model system, anantibody that recognizes a bacterial cell wall protein, the Moraxellacatarrhalis OMPE protein, was used. A bacterial recombinant fragment ofM. catarrhalis OMPE protein was provided by Dr. Timothy Murphy at theUniversity of Buffalo and used as the immunogen for preparing monoclonalantibody 12D.5 (Mab 12D.5). Using overlapping peptide mapping, theantigenic epitope was shown to reside within amino acids 187-220 of therecombinant fragment. A peptide corresponding to this epitope,designated Mopep2, was synthesized with an N-terminal cysteine forpost-synthesis attachment to free sulfhydryl-binding structures. Mab12D.5 was cleaved into two Fab fragments using 2-mercaptoethylamine HCL(Pierce) leaving a reactive sulfhydryl group that can be attached toanother molecule.

Ten milligrams per milliliter of magnetic particle stock Micromod,Nanomag Silica™, NH250, (lot number 210213T, catalogue number 13-01-252,Micromod Partikeltechnologie GmbH, Rostock-Warnemuende, Germany), whichcontains a free amino group on its surface, was reacted with 140micrograms SIAB in DMSO and rotated in the dark for 2 hours. Theparticles were separated by means of a magnet and subsequently incubatedin 50 millimolar sodium borate buffer pH 9.6 containing 5 millimolarEDTA with 100 microliter of 1.7 milligrams per milliliter Mopep2 in DMSOand incubated overnight. The free sulfhlydryl group on the Mopep2peptide was reacted with the SIAB-treated magnetic particles creating astable thioether bond. Unreacted sites were blocked with free cysteineand the Mopep2-labelled particles were washed and resuspended in 1milliliter PBS for analysis.

To test the ability of the Mopep2 particles to bind to free 12D.5 Mab insolution, an inhibition ELISA was performed. Ten microliters of eitherMopep2-particles or control particles similarly labelled with bovineserum albumin (BSA) were incubated with a 2-fold serial dilution ofhorseradish peroxidase (HRPO)-conjugated 12D.5 in PBS (total volume 100microliters) starting at 2 microgram/millilitre in a 96-well microtiterplate and incubated for 30 minutes. Particles were separated to thebottom of the wells by means of a magnet and 50 microliters of eachdilution set were added to Mopep2-coated plates and incubated for anadditional 30 minutes. A standard ELISA was then performed with theaddition of 3,3′5,5′-tetramethylbenzidine substrate, allowed to developfor 10 minutes, and absorbance read at 630 nanometers with no stopsolution added. Data depicted in FIG. 5 show that the Mopep2 particleswere capable of binding to and separating monoclonal antibody 12D5(12D.5 Mab) horseradish peroxidase (HRPO) conjugates from solution, thuspreventing the 12D.5 Mab HRPO conjugate from binding to Mopep2-coatedwells in a dose-dependent manner. Particles coated with bovine serumalbumin (BSA) were unable to inhibit 12D.5 Mab HRPO conjugate frombinding to Mopep2-coated wells.

Example III Preparation of the Binding Construct

This example provides one embodiment of the binding construct in whichan Fab fragment is used as the recognition portion of the bindingconstruct. The plasmid pUC 19 was purchased from Invitrogen and plasmidDNA was linearized by EcoRI. The linearized pUC19 DNA was extracted andpurified from agarose gel. This DNA was attached to the Fab fragment ofMab 12D.5 and served as the nucleic acid portion of the bindingconstruct. Commercially available primers were subsequently used toamplify a 1 kilobase fragment from the linearized pUC19 template. Togenerate a DNA fragment that would be attached to the 12D.5 Fab fragmentthrough the free sulfhydryls (—SH) and serve as the nucleic acid portionof the binding construct, a 5′-psoralen, 3′-amino oligonucleotide wasconjugated to a SIAB linker through the available amine. The pUC 1kilobase DNA was denatured in the presence of excess oligonucleotide andsnap annealed to the psoralen using long wave ultraviolet light. Theexcess oligonucleotide was removed with an molecular weight cut-off(MWCO) filter and the SIAB/pUC template was then attached to the Fabfragment using the free sulfhydryl-binding end of the attached linker.The entire binding construct, designated Fab-DNA, was not furtherpurified.

Example IV Detection of a Compound of Interest by PCR

This example provides one embodiment for detection of a compound ofinterest wherein the nucleic acid portion of the binding construct isamplified by PCR. This experiment demonstrated the ability of theFab-DNA binding construct (Mab 12D.5/pUC19 construct) to bind to thecompound of interest, the free antigen (bacterial recombinant fragmentof OMPE or rOMPE), thus forming construct-compound complexes insolution. Surfaces (magnetic particles) bearing accessible bindingtargets (Mopep2 peptides) bound any Fab-DNA not bound to the compound ofinterest, and a magnet was used to separate the surfaces, leaving theconstruct-compound complexes (Fab-DNA bound to antigen) in solution withthe nucleic acid portion (pUC19) of the binding construct available fornucleic acid amplification.

rOMPE was titered in two-fold dilution steps in 1 milliliter PBS from100 nanograms/millilitre to 1.56 nanograms/millilitre. Each serialdilution was incubated with 20 microliters (8 micrograms of Fab-DNA) for30 minutes, and then 10 microliters of Mopep2 particles was added toeach well and incubated for an additional 30 minutes. Control wellsincluded: (1) 1.1 milliliter PBS and 0.2 milliliter Fab-DNA no rOMPE(positive control), and (2) 1 milliliter PBS, 0.2 milliliter Fab-DNA,and 0.1 milliliter particles (negative control). Wells were exposed to arare earth magnet for 15 minutes to separate the magnetic particles fromthe solution, and 0.2 milliliter of the solution removed from each wellfor amplification.

Samples were amplified by PCR as follows. A 50 microliter PCR reactionmix contained 5 microliters 10×PCR buffer without MgCl₂, 1 microliter of50 millimolar MgCl₂, 0.5 microliter of 50 millimolar dNTPs, 1 microliterof each sense or antisense primer to pUC19, 1 microliter Taq polymerase,2 microliters of sample (solution), and 38.5 microliters ddH₂O. Thesense primer had the sequence CCTCTAGAGTCGACCTGCAGGCATGC (SEQ ID NO. 1).The anti-sense primer had the sequence CACTGGCCGTCGTTTFACAACGTCGTG (SEQID) NO. 2). All sequences are given in the 5′ to 3′ direction.

Samples were amplified using the following cycling parameters:

STEP TIME TEMPERATURE CYCLES Initial denaturation 2 minutes 93° C. 1Denaturation 30 seconds 93° C. 35 Annealing 30 seconds 52° C. 35Extension 2 minutes 73° C. 35 Final extension 7 minutes 73° C. 1

PCR samples were resolved on 1% agarose gel and visualized by ethidiumbromide. 10 microliters of each concentration of PCR samples was loadedon the gel. PCR primers amplified an approximate 2650 bp pUC19 fragment.A 1 kilobase extension DNA ladder (Invitrogen) was used as the molecularweight standard. As shown in FIG. 6, PCR primers amplified a 2.6kilobase DNA fragment in the following samples: 790 picogram, 395picogram, 198 picogram, 98 picogram, and 12 picogram. Very fine bandswere observed in samples of 49 picogram and 24 picogram.

Example V Detection of Compounds of the Interest

Non-limiting examples of detection of different types of compounds ofinterest follow.

Beta-Amyloid Protofibrils: In this example, the compound of interest isan oligomer of amyloid beta protein, called a protofibril. Detection ofamyloid beta protofibrils can be useful in the early detection ofAlzheimer's disease. The sample is serum or cerebral spinal fluid (CSF)from a patient suspected of having or being at risk for Alzheimer'sdisease. The binding construct includes (1) a recognition portion, whichis an antibody or an antibody fragment that recognizes the protofibril,and (2) a nucleic acid portion that does not exist in nature. Thenucleic acid portion is a DNA template that can be recognized bysynthetic primers designed to bind to the template at low stringency foramplification using PCR. The binding construct is added to aconcentration of 10 nanograms per milliliter with a 100 microlitersample of serum, and incubated for a sufficient time to allow theantibody or antibody fragment to bind to protofibrils present in thesample, thus forming construct-compound (construct-protofibril)complexes in solution. The binding surfaces are magnetic particleslabelled with an accessible binding target, the same peptide sequencethat was used as the immunogen to generate the anti-protofibril antibody(the recognition portion of the binding construct). The magneticparticles bear primary amine reactive groups, allowing the immunogenicpeptide to be conjugated to the particles through its carboxy-terminuscarboxyl using a water-soluble carbodiimide such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Ten microliters ofa 30% particle suspension is added to the solution containingconstruct-compound (construct-protofibril) complexes, allowing theparticles to form construct-surface complexes with the unboundanti-protofibril/DNA binding constructs. A magnet is applied to thesolution and the magnetic particles are separated, thus separatingunbound binding constructs and leaving behind the construct-compoundcomplexes in the solution. Two microliters of the resulting solution(containing construct-compound complexes) are added to a PCR reactionsolution containing primers, free nucleotides, and Taq DNA polymerase.The DNA portion of any binding constructs present as construct-compoundcomplexes in solution is amplified. The PCR reaction is resolved on a 1%agarose gel and visualized by ethidium bromide staining. The presence orabsence of amyloid beta protofibrils in the sample is indicated by thepresence or absence, respectively, in the gel of bands corresponding tothe correct molecular weight for the amplified DNA fragment.Legionella pneumophilia Antigen: In this example, the compound ofinterest is a Legionella pneumophilia serogroup 1 carbohydrate antigen.L. pneumophilia is the causative agent of the community acquiredpneumonia known as Legionnaire's disease, and of Pontiac fever.Serogroup 1 is responsible for the majority of cases of Legionnaire'sdisease. The sample is preferably a urine sample from a patientsuspected of having a L. pneumophilia infection. The binding constructincludes (1) a recognition portion, which is an antibody or an antibodyfragment that recognizes the L. pneumophilia serogroup 1 carbohydrateantigen (“antigen”), and (2) a nucleic acid portion that does not existin nature. The nucleic acid portion is a DNA template that can berecognized by synthetic primers designed to bind to the template at lowstringency for amplification using PCR. The binding construct is addedto a concentration of 10 nanograms per milliliter with a 100 microlitersample of urine, and incubated for a sufficient time to allow theantibody or antibody fragment to bind to L. pneumophilia serogroup 1carbohydrate antigen present in the sample, thus formingconstruct-compound (construct-antigen) complexes in solution. Thebinding surfaces are magnetic particles labelled with an accessiblebinding target, a peptide mimotope that is capable of binding to thevariable regions of the antibody or antibody fragment (the recognitionportion of the binding construct). A suitable peptide mimotope can beobtained by methods known in the art, such as phage display of a randomor non-random peptide library or combinatorial peptide synthesis,followed by affinity selection (Smith & Petrenko (1997) Chem. Rev.,97:391-410; Kramer et al. (1993) Peptide Res., 6:314-319). The magneticparticles bear primary amine reactive groups, allowing the peptidemimotope to be conjugated to the particles through its carboxy-terminuscarboxyl using a water-soluble carbodiimide such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Ten microliters ofa 30% particle suspension is added to the solution containingconstruct-compound (construct-L. pneumophilia serogroup 1 carbohydrateantigen) complexes, allowing the particles to form construct-surfacecomplexes with the unbound anti-antigen/DNA binding constructs. A magnetis applied to the solution and the magnetic particles are separated,thus separating unbound binding constructs and leaving behind theconstruct-compound complexes in the solution. Two microliters of theresulting solution (containing construct-compound complexes) are addedto a PCR reaction solution containing primers, free nucleotides, and TaqDNA polymerase. The DNA portion of any binding constructs present asconstruct-compound complexes in solution is amplified. The PCR reactionis resolved on a 1% agarose gel and visualized by ethidium bromidestaining. The presence or absence of L. pneumophilia serogroup 1carbohydrate antigen in the sample is indicated by the presence orabsence, respectively, in the gel of bands corresponding to the correctmolecular weight for the amplified DNA fragment.Mycobacterium tuberculosis-specific Human Antibodies: In this example,the compound of interest is Mycobacterium tuberculosis-specific humanIgG specific for a 38 kilodalton extracellular protein from M.tuberculosis (anti-MTh IgG). M. tuberculosis is the causative agent ofpulmonary tuberculosis in humans. The sample is preferably serum from apatient suspected of having a M. tuberculosis infection. The bindingconstruct includes: (1) a recognition portion, which is a peptideepitope or a mimotope that is recognized and bound by the variableregion of the anti-MTh IgG, and (2) a nucleic acid portion, mostpreferably a nucleic acid portion wherein the sequence of the nucleicacid portion does not include a sequence that is expected to be found inthe sample, for example a nucleic acid sequence from higher plants thatis not found in either mammals or bacteria. The recognition portion canbe a mimotope for the anti-MTb IgG or a native peptide sequence derivedfrom the bacterial protein or peptide that is recognized by the anti-MThIgG; in either case, the peptide is capable of binding to a subclass ofM. tuberculosis-specific human immunoglobulin G and formingconstruct-compound (construct-anti-MTh IgG) complexes. The nucleic acidportion is a DNA template that can be recognized by synthetic primersdesigned to bind to the template at low stringency for amplificationusing PCR. The binding construct is added to a concentration of 10nanograms per milliliter with a 100 microliter sample of serum, andincubated for a sufficient time to allow the recognition portion of thebinding construct to bind to anti-MTh IgG present in the sample, thusforming construct-compound (construct-anti-MTh IgG) complexes insolution. The binding surfaces are agarose beads to which are attachedan antibody or antibody fragment that is capable of recognizing andbinding to the recognition portion of the binding construct. The agarosebeads bear aldehyde reactive groups which may be crosslinked to an aminogroup on the antibody's heavy chain using sodium cyanoborohydride(NaBH₃CN). Twenty microliters of a 30% particle suspension is added tothe solution containing construct-compound (construct-anti-MTh IgG)complexes, allowing the beads to form construct-surface complexes withthe unbound peptide/DNA binding constructs. The mixture is placed in anEppendorf tube and centrifuged to pellet the agarose beads out ofsuspension, leaving the construct-compound (construct-anti-MTh IgG)complexes remaining free in solution in the supernatant. Two microlitersof the supernatant solution are added to a PCR reaction solutioncontaining primers, free nucleotides, and Taq DNA polymerase. The DNAportion of any binding constructs present as construct-compoundcomplexes in solution is amplified. The PCR reaction is resolved on a 1%agarose gel and visualized by ethidium bromide staining. The presence orabsence of Mycobacterium tuberculosis-specific human immunoglobulin G inthe sample is indicated by the presence or absence, respectively, in thegel of bands corresponding to the correct molecular weight for theamplified DNA fragment.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified. Various changes and departures may be made to the presentinvention without departing from the spirit and scope thereof.Accordingly, it is not intended that the invention be limited to thatspecifically described in the specification or as illustrated in thedrawings, but only as set forth in the claims.

1. A method for detecting a non-nucleic acid compound of interest in abiological or environmental sample comprising the steps of: a) providinga binding construct comprising an antibody or an antibody fragmentportion which specifically recognizes and specifically binds saidnon-nucleic acid compound of interest without capture on a solidsupport, and a nucleic acid portion; b) mixing said binding constructwith said biological or environmental sample in a solution mixture toform construct-compound complexes in solution without capture on a solidsupport, wherein said non-nucleic acid compound of interest in saidbiological or environmental sample becomes specifically bound to saidbinding construct; c) providing one or more surfaces, wherein saidsurfaces bear one or more accessible non-nucleic acid binding targetscapable of specifically recognizing and specifically binding to saidantibody or said antibody fragment portion of said binding construct; d)introducing said one or more surfaces to said solution mixture of saidconstruct-compound complexes after said non-nucleic acid compound ofinterest in said biological or environmental sample has become bound tosaid binding construct in order for said one or more surfaces to formconstruct-surface complexes in solution with any unbound bindingconstructs resulting in said solution mixture containing essentiallysaid construct-compound complexes and said construct-surface complexes;e) separating said construct-surface complexes from said solutionmixture leaving behind said construct-compound complexes in solution;and f) detecting the presence or absence of said nucleic acid portion ofsaid construct-compound complexes in solution after said separation step(e), said detection being done without the presence of any of saidsurfaces in connection with said construct-compound complexes that arebeing detected; wherein the presence of said nucleic acid portion ofsaid construct-compound complexes indicates the presence of saidnon-nucleic acid compound of interest in said biological orenvironmental sample.
 2. The method of claim 1, wherein said one or moresurfaces is selected from the group consisting of: particles, powders,beads, planar surfaces, non-planar surfaces, a tube, a well, non-porousfilms, non-porous membranes, porous films, porous membranes, fibers,fillers, meshes, grids, filters, matrices, gels, and combinationsthereof.
 3. The method of claim 1, wherein said one or more surfacescomprise particles.
 4. The method of claim 3, wherein said particlescomprise magnetic particles.
 5. The method of claim 4, wherein said step(e) comprises separating said construct-surface complexes out of saidmixture by means of a magnet.
 6. The method of claim 5, wherein, in step(f), said detecting the presence or absence of said nucleic acid portionof said binding construct comprises amplification of said nucleic acidportion.
 7. The method of claim 6, wherein said amplification of saidnucleic acid portion comprises a polymerase chain reaction.
 8. Themethod of claim 1, wherein, in step (f), said detecting the presence orabsence of said nucleic acid portion of said binding construct comprisesamplification of said nucleic acid portion.
 9. The method of claim 8,wherein said amplification of said nucleic acid portion comprises apolymerase chain reaction.
 10. The method of claim 1, wherein saidantibody or antibody fragment portion comprises a single chain antibodyvariable region fragment.
 11. The method of claim 1, wherein saidantibody or antibody fragment portion comprises a Fab fragment.
 12. Themethod of claim 11, wherein said Fab fragment is attached to saidnucleic acid portion through the free sulfhydryl of the Fab fragment.13. The method of claim 1, wherein said nucleic acid portion comprisesDNA.
 14. The method of claim 1, wherein said nucleic acid portioncomprises RNA.
 15. The method of claim 1, wherein said nucleic acidportion comprises a nucleic sequence that does not include a sequencethat is expected to be found in the biological or environmental sample.16. The method of claim 1, wherein said step (a) comprises providing twoor more different types of binding constructs, wherein each of said twoor more different binding constructs has a different antibody orantibody fragment portion and a different nucleic acid portion.
 17. Amethod for solution-phase detection of a non-nucleic acid compound ofinterest, comprising the steps of: a) providing a biological orenvironmental sample suspected of containing said non-nucleic acidcompound of interest; b) providing a binding construct comprising: i) anantibody or an antibody fragment portion capable of specifically bindingsaid non-nucleic acid compound of interest without capture on a solidsupport, and ii) a nucleic acid portion c) contacting said biological orenvironmental sample with said binding construct for a period of timesufficient to permit said antibody or antibody fragment portion tospecifically bind said non-nucleic acid compound of interest present insaid biological or environmental sample, thereby formingconstruct-compound complexes in solution, wherein said non-nucleic acidcompound of interest in said biological or environmental sample becomesspecifically bound to said binding construct; d) providing one or moresurfaces, wherein said one or more surfaces bears one or more accessiblenon-nucleic acid binding targets capable of specifically binding to saidantibody or antibody fragment portion; e) contacting said one or moresurfaces with said solution alter said non-nucleic acid compound ofinterest in said biological or environmental sample has become bound tosaid binding construct for a period of time sufficient for said one ormore accessible non-nucleic acid binding targets to specifically bindsaid antibody or antibody fragment portion of any binding construct notbound to said non-nucleic acid compound of interest, thereby formingconstruct-surface complexes in said solution resulting in said solutioncontaining essentially said construct-compound complexes and saidconstruct-surface complexes; f) separating said construct-surfacecomplexes from said solution, leaving said construct-compound complexesin said solution; and g) detecting the presence or absence of saidnucleic acid portion of said construct-compound complexes in saidsolution after said separation step (f), said detection being donewithout the presence of any said surfaces in connection with saidconstruct-compound complexes that are being detected; wherein saidseparation of said construct-surface complexes from said solutionresults in a separation of all binding constructs not bound to anon-nucleic acid compound of interest, and wherein the presence of saidnucleic acid portion of said construct-compound complexes indicates thepresence of said non-nucleic acid compound of interest in saidbiological or environmental sample.
 18. The method of claim 17, whereinsaid one or more surfaces is selected from the group consisting of:particles, powders, beads, planar surfaces, non-planar surfaces, a tube,a well, non-porous films, non-porous membranes, porous films, porousmembranes, fibers, fillers, meshes, grids, filters, matrices, gels, andcombinations thereof.
 19. The method of claim 17, wherein said one ormore surfaces comprise particles.
 20. The method of claim 19, whereinsaid particles comprise magnetic particles.
 21. The method of claim 20,wherein said step (f) comprises separating substantially all saidconstruct-surface complexes from said solution by means of a magnet. 22.The method of claim 21, wherein, in step (g), said detecting thepresence or absence of said nucleic acid portion of said bindingconstruct comprises amplification of said nucleic acid portion.
 23. Themethod of claim 22, wherein said amplification of said nucleic acidportion comprises a polymerase chain reaction.
 24. The method of claim17, wherein, in step (g), said detecting the presence or absence of saidnucleic acid portion of said binding construct comprises amplificationof said nucleic acid portion.
 25. The method of claim 24, wherein saidamplification of said nucleic acid portion comprises a polymerase chainreaction.
 26. The method of claim 17, wherein said antibody or antibodyfragment portion comprises a single chain antibody variable regionfragment.
 27. The method of claim 17, wherein said antibody or antibodyfragment portion comprises a Fab fragment.
 28. The method of claim 27,wherein said Fab fragment is attached to said nucleic acid portionthrough the free sulfhydryl of the Fab fragment.
 29. The method of claim17, wherein said nucleic acid portion comprises DNA.
 30. The method ofclaim 17, wherein said nucleic acid portion comprises RNA.
 31. Themethod of claim 17, wherein said nucleic acid portion comprises anucleic sequence that does not include a sequence that is expected to befound in said biological or environmental sample.
 32. The method ofclaim 17, wherein said step (b) comprises providing two or moredifferent types of binding constructs, wherein each of said two or moredifferent binding constructs has a different antibody or antibodyfragment portion and a different nucleic acid portion.